Substance possessing antimicrobial activity and disease control activity on some plant diseases and the method for its production



Nov. 10, 196 HACHIRO OKU ETAL 3,156,618

SUBSTANCE POSSESSING ANTIMICROBIAL ACTIVITY AND DISEASE CONTROL ACTIVITYON SOME PLANT DISEASES AND THE METHOD FOR ITS PRODUCTION Filed Jan. 24,1963 2 Sheets-Sheet 1 Wave/en; 2%

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1964 HACHIRO OKU ETAL 3,156,618

SUBSTANCE POSSESSING ANTIMICROBIAL ACTIVITY AND DISEASE CONTROL ACTIVITYON SOME PLANT DISEASES AND THE METHOD FOR ITS PRODUCTION Filed Jan. 24,1963 2 Sheets-Sheet. 2

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United States Patent 3,156,618 SUBSTANCE ?OSESSING ANTill HCROBIAL AC-TIVITY AND DISEASE (IGNTRGL ACTIVITY ON SOME PLANT DISEASES AND THEMETHGD FQR ITS PRUDUCTION Hashim (ll tn and Toshiro Nakanlshi, Tokyo,Japan, assignors to Sanlryo Company, Limited, Tolryo, lapan Filed Jan.24, 1963, Ser. No. 253,721 Claims priority, application Japan, Feb. 8,H62, 37/3,94t 2 Claims. (Cl. 167-65) This invention relates to a new anduseful substance possessing antimicrobial activity and disease controlactivity on some plant diseases, and to the method for its production.More particularly, it relates to a new and useful antibiotic calledascochytin and to the method for production of the antibiotic.

It is an object of this invention to provide a new and usefulantibiotic, ascochytin.

Another object of this invention is to provide method of producing thisantibiotic, ascochy-tin, by microbiological processes.

Physical and chemical properties of ascochytin are shown below.

PHYSICAL AND CHEMlCAL PROPERTIES OF ASCOCl-IYTIN Ascochytin is aslightly acidic yellow needle crystalline substance and melts at 200-201C. with decomposition. The analytical data are as follows:

Calcd for C H O C, 65.21; H, 5.84; O, 28.95. IVLW. 276.28. Found: C,65.66; H, 5.91; O, 28.43. M.W. 284.54 (measured by osmorneter).

It contains no nitrogen, halogen, sulfur or phosphorus.

The degree of specific rotation of ascochytin is [a] -85.99 (c.=1 inchloroform).

The visible and ultraviolet absorption spectrum is shown in FIG. 1. Ithas two characteristic absorption maxima at 286 mu and at 4-15 m in anethanol solution. The infrared absorption spectrum of ascochytin isshown in FIG. 2. It exhibits characteristic absorption hands in theinfrared region of the spectrum, in a potassium-bromide pellet, at thefollowing frequencies expressed in reciprocal centimeters: 3100, 2950,1640, 1590, 1530, 1460, 1410, 1340, 1250, 1200, 1160, 1120, 107"), 1050,1005, 960, 935,

and 835.

Ascochytin is very soluble in chloroform, moderately in methanol,ethanol, acetone, ethylacetate, ethylether, benzene, ethylenglycol, andsparingly soluble in n-hexane and petroleum other, but almost insolublein water. It .gives a strong greenish fluorescence in ethanol solutionunder the ultraviolet ray.

Its yellow color in ethanol is decolorized by addition of magnesiumacetate, one drop of N NaGH or hydrosulfite. Potassium permanganate isdeoolorized. It gives a red ferric-chloride reaction in ethanol and doesnot reduce Fehlings solution.

THE MICRGORGANISM The method forproduction of ascochytin by micro-3,l5h,hl3 Patented Nov. 10, 1964:

THE ANTIBIOTIC The production of ascochytin in accordance with thepresent invention is carried out by inoculating a sterile aqueousnutrient medium with Ascochyta fabae Spegazzini, incubating theincoculated medium under aseptic aerobic conditions at a temperaturebetween about 23-30 C., and isolating the desired ascochytin from thesolid material present in the culture mixture and the aqueous cultureliquid.

For the inoculation, spores and/or mycelium of Asc0- zym fubaeSpegazzini can be used. A piece of mycelium is sumcient-ly used as theinoculum. For large scale of fermentations it is preferable to usevigorous, young cultures of the microorganism.

Suitable aqueous nutrient media are those having a pH between 5 and 10,preferably between 6.5-9.-0, and containing an assimilable carbonsource, a source of nitrogen and minerals. As the assimilable carbonsources, either pure carbohydrates or commercially availablecarbohydrate mixtures may be used. Some examples of the materials whichare suitable for this purpose include glucose, mannose, lactose,sucrose, maltose, Xylose, solule starch and the like. The quantity ofthe carbohydrates present in the nutrient medium is not particularlycritical and can vary from about 0.5% to 8% by weight of the totalweight of the medium.

The source of nitrogen in the nutrient medium may be of an organic,inorganic, or mixed organic-inorganic nature. Some examples of the manynitrogenous substances Which ray be employed in the nutrient medium arepep-tones, amino acids, hydrolyzed and unhydrolyzed proteins, soybeanmeal, corn steep liquor, inorganic nitrates, urea, ammonium salts andthe like. Due to the crude nature or most of the readily availablenitrogenous substances, the quantity to be added to the nutrient mediumvaries somewhat in accordance with purity. However, it can be said forpractical purposes that nitrogenous materials need not exceed 4% byweight of the total weight of the fermentation medium.

A certain amount of mineral salts and vita-mines are necessary to obtainthe best yields of ascochytin. In general, it is usually advantageous toadd a small amount of inorganic salts, such as phosphate, ferrous salts,potassium salts, magnesium salt and so on. As to vitamins, althoughaddition of small amount of Vitamin B biotin, Ca-pantothenate and thelike is advantageous, they are not always necessary for the productionof ascochytin because the other crude materials to be added into thenutrient medium contain fairly sufiicient amounts of these substances.

It is because the yields of the'antibiotic ascochytin is considerablyreduced by the operation beyond the scope of pl-lbetween 5.0 and 10 thatthe aforementioned scope has been specified in the present invention.

The cultivation of A'scochyta fabae Spegazzini in the aqueous nutrientmedium can be carried out in a number of di'llerent ways. For example,the microorganism can be cultivated under aerobic conditions on thesurface of the medium or it can be cultivated beneath the surface of themedium, that is, in the submerged condition, if oxygen is simultaneouslysupplied.

The preferred method, for producing ascochytin on a large scale involvesthe use ofsubmerged'or deep cultures of Ascochytzz fabzze Spegazzini.According to this The length of time required for the maximum productionof ascochytin varies with the size and type of equipment used. Forexample, in large-scale commercial fermentation such as one carried outin the tank-type fermentors, maximum production of ascochytin is reachedin about four to ten days. Incubation time varies with the incubationmethod of inoculation and the like. Therefore, longer period ofincubation does not reduce the yield of ascochytin suddenly, unlessotherwise the pH of culture medium shifts beyond the scope between 5 and10. Under submerged culture conditions, the microorganism develops asmore or less discrete particles dispersed throughout the nutrient mediumin contrast to the more or less continuous pellicle present on thesurface of the medium in the surface culture method. By virtue of thisdistribution of the organism throughout the medium, large volumes of theinoculated nutrient medium can be cultivated at one time in the largetanks and vats customarily employed in the fermentation industry.Stationary vat fermentors equipped with suitable agitation and aerationdevices as well as horizontal rotary drum fermentors have been found tobe particularly useful in this respect. However, for the preparation ofsmaller quantities of the antibiotic or of cultures of themicroorganism, the submerged culture method may be carried out in smallflasks or jars which are either shaken or stirred by suitable mechanicalmeans.

Agitation and aeration of the culture mixture may be accomplished in anumber of ways. Agitation may be provided by turbines, paddles,impellers or other mechanical agitation devices, by revolving or shakingthe fermentor itself, by various pumping devises or by the passage ofair through the medium. Aeration may be effected by injecting air intothe fermentation mixture through open pipes, perforated pipes, porousdiffusion media such as carbon sticks, Carborundum, sintered glass andthe like, or it may be provided by spraying, splashing or spilling themash into or through oxygen-containing atmosphere.

In order to isolate the effective substance present in the culturemixture after completion of the fermentation phase of the process, theconventional means usually used for the isolation of effective compoundscontained in various fermentation mixture may be conveniently employed.On isolating ascochytin from the above-described sources, such means asconcentration of the effective substance, removal of impurities andconversion to composition capable of being more readily purified areused in accordance with and by the use of the properties of ascochytin.Difference in solubilities in various solvent from the other impuritiescontained in culture mixture, partition coefficients into two liquidlayers, absorption afiinities, and degree of dissociation of hydrogenion are the means being made use of. As the embodied operations applyingthese means are involved, for example, dissolution or extraction withsolvents, and precipitation from solvents.

The methods of isolation of ascochytin by the use of aforementionedmeans will be in details described below. These detailed method are,however, to be understood as illustrative but not limited to it.

Upon treating the liquid culture mixture containing mycelium it ispreferable to carry out extraction by the use of solvents which dissolveascochytin but are immiscible with water, such as for example,chloroform, ethyl acetate and so on. It is desirable to conduct theextraction at a pH between 3.0 and 5.0 but pH below or higher than theabove may be applicable.

Upon subjecting the solid material containing mycelium and the culturefiltrate separately to isolation procedures, ascochytin in the liquidphase can be separated from impurities by the use of difference inpartition coefficient between aqueous and organic-solvent layers in thesame way as described above, and the antibiotic present in the solidmaterial containing mycelium can be extracted 4. with suitable solventscapable of dissolving the antibiotic such as methanol, ethanol, acetone,chloroform, acetic acid esters and the like.

When the culture liquid containing or without mycelium is concentratedor solidified by vacuum distillation or spray drying, ascochytin can beisolated in the same way as described in the aforementioned case ofsolid material containing mycelium. If the degree of concentration islow the treatment is effected in the same way as described aboveregarding the case of liquid culture mixture. Ascochytin dissolved inthe solvent can be isolated by removing the solvent by such means asvacuum distillation. Alternatively, it may be isolated by precipitatingit following addition of a solvent in which it is sparingly soluble,such as for example, petroleum ether or water, to the solution. Furtheralternativeiy, the effective substance may be isolated by removing theimpurities by washing the residue with solvent in which aschochytin isalmost insoluble after removing the solvent in which it is dissolved.

Ascochytin thus obtained is readily crystallized as the crude crystalswhen allowed to stand, preferably at low temperatures, after removal ofthe solvent to a suitable egree. The crude ascochytin can be purified bytreating it with various organic solvents, a solvent or solvent mixturessuch as, for example, methanol, ethanol, acetone, benzene and the likethrough procedures such as concentration, standing and cooling.

In view of the aforementioned properties of ascochytin it is conceivablethat the substance obtained by the invention is a new one and may beeasily isolated.

Examples will be shown below where combinations of plural methods ofisolation are shown. However it will be understood from the descriptionsabove that by each single method of isolation also may be carried theprocess according to this invention. Now that properties of theeffective substance are disclosed as above it will be evident by thoseskilled in the art that the object or" this invention can be attainedalso by other alternations or modified methods which are not concretelydescribed in the specification of the present invention.

BIOLOGICAL PROPERTIES OF ASCOCHYTIN Antimicrobial spectrum of ascochytinwas examined by two fold agar dilution method for a period of 1 4 days.The antibiotic was dissolved in a small amount of dimethylformamide anddiluted with sterilized water to give a final agar medium containingless than one percent of the solvent, which was not a harmfulconcentration against the organisms tested. Studies with animalparasitic bacteria were conducted in a Bacto Nutrient Agar (which ismanufactured and sold by Difco Lab). For plant pathogcnic bacteria andfungi potato-sucrose agar medium was used. Yeasts and Trichophytons weretested in Sabouraud medium. The results are shown in Table l.

Ascochytin is found to have a very broad spectrum. It is effectiveagainst some plant pathogenic bacteria, yeasts, and fungi.

Table ].Antimicrobial Spectrum of Ascochytin Test organism Minimuminhibitory X anthomonas oryzae Bacterium pruni Erwinia aroz'deae 100Yeasts:

Zygosaccharomyces sulsus 50 S accharomyces cerevisiae 100 Candidaalbz'ca'ns 50 Table lContinued Fungi:

Pirictqiaria oryzae 1.5 3 Glomerella cingulata 3 Cochliobolus miyabeanus100 A lternaria kikuchiana 50 Gloeosporiam kaki 12.5 Gloeosporiumlaeticol or 50 Gibberella saubinetii 12.5 Gibberella fujikuroi 100Fusarium nivennm 100 F usarium lycopersici 10 Fusarium lini 100Aspergillus niger 100 Aspergillas oryzae 100 Penicillz'um digitatum 2Penicillium italicum 0 Trichophyton interdigitale 50 T rich ophyton rubrzzm 5 0 T rz' chophy ton asteroid es 5 0 Table 2.-Inlzibitl0h of SporeGermination in Some Plant Pathogenic Fungi by Ascochytin Percentgermination (Joncentration of ascoehytin (meg/ml.) Pirz'cularlaCochliabolus oryzce miyabeanus As is shown in the above table,'ascochytin completely inhibits the spore germination of Piriculariaoryzae at the concentration of 6 ppm. and of Cochliobolns miyabeanus at25 ppm.

Ascochytin is found to be a valuable substance for control of some plantdiseases. 1

The protective effect of a-scochytin against blast disease of rice plant(Oryza sativa L.) was found out by the following examinations. The youngseedling of rice plants (the variety Shigaasahi No. 27) grown in smallpots for about 4 weeks were sprayed with 10 ml. of aqueous suspension ofwe-ttable powder of asc-ochytin per pot. The rascochytin concentrationof sprayed solution was 1000 ppm. After drying, the test plants wereinoculated with a conidial suspension of blast fungus, Piriculariaoryzae Cav., and kept in a moist chamber (97% relative humidity) at 23C. for 48 hours. Untreated plants were also inoculated simultaneously asthe check. The inocu la'tedpl-ant-s are then transferred into agreenhouse. At 3 days after inoculation, the diseased degree wasdetermined on treated and check plan-ts as the percentage of diseasedleaves.

Table 3.Pr0tective Efiect of Ascochytin Against blast Disease of RicePlant As is shown in Table 3, the susceptibility of rice plant to blastdisease was markedly reduced by treatment with ascochytin at theconcentration of 1000 p.p.m.

The protective effect of ascochytin against l-ate blight disease oftomato was examined by two days, namely by detached leaf method and byinoculation test in greenhouse.

The detached leaf method was carried out as follows: A suspension ofwettable powder of ascochytin was sprayed on newly cut tomato leaves.The con-tent of ascochytin in the sprayed solution was 1000 ppm. Afterdrying, fine treated leaves were put into a 15 cm. petri dish andinoculated with a drop of spore suspension of late blight fungus,Phytophthora infestans de Bary. Then,

the inoculated leaves were kept in a controlled chamber of 20 C.temperature and 100% relative humidity. As the check, untreated leaveswere inoculated simultaneously. At 5 days after inoculation, diseaseddegree of the cut leaves was determined. Diseased degree was estimatedas follows:

The symptoms of tomato leaves were divided into 6 grades according tothe size of lesions, for example, healthy leaf was marked as 0, and thelargest lesion as 5. The effect of ascochytin treatment was expressed asthe percentage of total marks of lesions in treated leaves to that ofuntreated ones.

Table 4 .-Protective E fleet of Ascochytin Against Tomato Late BlightDisease Examined by Detached Lead The inoculation test of tomato lateblight disease in greenhouse was conducted as follows: Tomato seedlings(the variety Shinfukuju) grown in the greenhouse tor a month weresprayed with the suspension of ascochytin wettable powder which contains1000 ppm. ascochytin. After 24 hours, the treated and untreated checktomato seedings were inoculated with a spore suspension of late blightfungus of tomato and were incubated for 24 hours in a moist chamber at20-23 (3., and then, they were transferred into the greenhouse. Theresult of observation at 3 days after inoculation is shown in Table 5.The method of estimation of lesion is the same as in the decached leaftest described above.

Table 5.-Protective Efiect of Ascochytin Against Tomato Late BlightDisease Examined by Greenhouse-Inoculation Test Untreated tomatoseedlings were entirely withered at 5 days after inoculation, while theascochytin treated tomatoes were still alive.

From the results of aforementioned experiments it was found thatascochytin is a very effective protect'tnt in agricultural use not onlyagainst blast disease of rice plant but also against tomato late blightdisease. In the above experiments, no phytotoxicity was observed.

Ascochytin may be used in suitable forms, such as for example, powderymixture with inactive diluent, emulsifiable solution with organicsolvent and compatible surface active agents, wettable powder withinactive diluent and adequate surface active agents, and granule withinactive diluent and small amount of water. In such cases, ascochytinmay be conveniently used in concentration between 0.1 and percent of thepreparations. Ascochytin may be employed, if desired, in combinationwith other antimicrobial or plant chemotherapeutic agents.

The following examples illustrate suitable methods for preparing,purifying and fractionating aschochytin.

EXAMPLE 1 One hundred ml. portions of a medium containing 2 g. ofglucose, 0.5 g. of peptone, 0.1 g. of dibasic potassium phosphate, 0.5g. of potassium chloride, 0.05 g. of magnesium sulfate, 0.001 g. offerrous sulfate, 03 mg. of thiamine, 0.1 mg. of vitamin B 0.1 mg. ofvitamin B and 0.3 of biotin are placed into each of one hundred of 500ml.=shakiug fermentation bottles in the conventional way. Aftersterilization, one loopful of mycelium of Ascoclzyta fabae Spegazzinicultivated on potato-sucrose agar slant is inoculated and the inoculatedbottles are incubated with shaking at 26 C. for 8 days. The productionof ascochytin then reaches to between and 7/ ml. in the broth.

The culture media are collected and the mycelium is separated byfiltration. The wet mycelium weighs 500 g. and is subjected toextraction with 2.5 kg. of acetone. The solvent is distilled off invacuum and the remaining rich water is extracted with chloroform. Thischloroform solution is treated by the same way as the case of chloroformextract prepared from the culture filtrate.

The culture filtrate, 7.5 1. in volume, is acidified with N-i-ICl to pH3-4, and is extracted with 1; volume of chloroform. The extract isconcentrated to remain a gummy brown substance. This substance isdissolved in hot n-hexane by repeated extractions, and then the hexaneis distilled off under reduced pressure. The remaining yellow solidsubstance is crystallized from hot ethanol. Recrystallization fromethanol, benzene or acetone gave a light yellow fine needle or plate.Pure ascochytin thus obtained is weighing 350 mg.

EXAMPLE 2 Three hundred liters of nutrient liquid containing 2% ofglucose, 0.25% of peptone, 0.2% of dibasic potassium phosphate, 0.05% ofpotassium chloride, 0.1% of magnesium sulfate and 0.001% of ferroussulfate is placed in a 600 1.:fermentation tank. The medium issterilized under pressure by the conventionalmethod and inoculated with3 l. of shake culture of Ascoclzyta fabae Spegazzini (cultured at 26 C.for 4 days). The inoculated medium is incubated at 26:1" C. withaeration of 300 l. per minute and agitation of 250 r.p.m. After 146hours of incubation 280 1. of culture liquid (including mycelium)containing 40 7/ cc. of ascochytin is obtained.

EXAMPLE 3 Two hundred and eighty liters of culture liquid of Ascochytafabae Spegazzini (containing 11.2 g. of ascochytin) obtained in the sameway as in Example 2 is acidified to pH 3.4 with hydrochloric acid and140 l. of chloroform is added to the acidified liquid. The mixture isagitated and separated by a centrifuge into 3 parts,

8) Waste broth, lnycelium and chloroform layer. The waste broth andmycelium are discarded because of their low content of ascochytin. Thechloroform extract is concentrated in vacuum to give a gummy substance.This gummy substance is dissolved in hot n-hcxane by repeatedextractions until no color is given in the extract. The solution isconcentrated in vacuum to leave a yellow solid substance. This solidsubstance is dissolved in 500 ml. of hot ethanol and allowed to standovernight in a refrigerator and the yellow crystals formed are collectedand dried to obtain 9.5 g. of crude ascochytin of purity.

EXAMPLE 4 Two hundred and eighty liters of the culture liquid obtainedin the same way as in Example 2 is adjusted to pH 3.4 with hydrochloricacid and l. of chloroform is added to the acidified liquid. After themixture is agitated vigorously, the chloroform layer is separated bycentrifugation, and concentrated in vacuum to about 3 1. Thisconcentrated solution is passed through a column of dibasic calciumphosphate. The black impurities are adsorbed in the upper part of thecolumn, and ascochytin is contained in the efiluent. The column isfurther washed with chloroform until to give a colorless efiiuent. Theellluent thus obtained is concentrated in vacuum to dryness and theremaining solid substance is crystalized from ethanol. The crudeascochytin obtained in this method is 9.3 g. (purity 85%). Five grams ofcrude ascochytin are dissolved in 200 ml. of boiling ethanol. Insolublematter is filtered off and the filtrate is allowed to stand inrefrigerator for overnight. The resulting crystals are collected andrecrystallized 3 times in the same way described above. The pure crystalof ascochytin thus obtained is 2.3 g. of light yellow fine needle andmelted at 200-201 C. with decomposition.

We claim:

1. Ascochytin, a substance being a slightly acidic, light yellow needlecrystal; containing the elements carbon, hydrogen and oxygen; being verysoluble in chloroform, moderately soluble in methanol, ethanol, acetone,ethylacetate, ethylether, benzene, ethyleneglycol, sparingly soluble inn-hexane and petroleum ether, and almost insoluble in water; giving astrong greenish fluorescence in solution under ultra-violet ray, givinga red color with ferric chloride reagent in ethanol, decolorizingpotassium permanganate, giving negative Fehlings test, being decolorizedwith magnesium acetate, sodium hydroxide and hydrosulfite; melting at atemperature in the range of 200-201 C. with decomposition; having adegree of optical rotation of 85.99 (1% in chloroform); havinganalytical data: C, 65.66; H, 5.91; O, 28.43; having visible andultra-violet absorption spectra in ethanol solution exhibiting twomaxima at 415 and 286 m and exhibiting characteristic absorptions in theinfrared region on the spectrum in potassium bromide pellet, at thefollowing frequencies expressed in reciprocal centimeters: 3100, 2950,1640, 1590, 1530, 1460, 1410, 1340, 1250, 1200, 1160, 1120, 1070, 1050,1005, 960, 935, and 835.

2. A process for the production of ascochytin which comprisescultivating Ascochyta fabae Spegazzini under aerobic conditions at atemperature in the range of from 20 to 32 C. in an aqueous nutrientmedium having a pH between 5 and 10 and containing an assimilable carbonsource, a source of nitrogen and minerals, and recovering ascochytin soproduced from the medium.

References Cited in the file of this patent Ann. Phytopath. Soc. Japan,17, (1953).

1. ASCOCHYTIN, A SUBSTANCE BEING A SLIGHTLY ACIDIC, LIGHT YELLOW NEEDLE CRYSTAL; CONTAINING THE ELEMENTS CARBON, HYDROGEN AND OXYGEN; BEING VERY SOLUBLE IN CHLOROFORM, MODERATELY SOLUBLE IN METHANOL, ETHANOL, ACETONE, ETHYLACETATE, ETHYLETHER, BENZENE, ETHYLENEGLYCOL, SPARINGLY SOLUBLE IN N-HEXANE AND PETROLEUM ETHER, AND ALMOST INSOLUBLE IN WATER; GIVING A STRONG GREENISH FLUORESCENCE IN SOLUTION UNDER ULTRA-VIOLET RAY, GIVING A RED COLOR WITH FERRIC CHLORIDE REAGENT IN ETHANOL, DECOLORIZING POTASSIUM PERMANGANATE, GIVING NEGATIVE FEHLING''S TEST, BEING DECOLORIZED WITH MAGNESIUM ACETATE, SODIUM HYDROXIDE AND HYDROSULFITE; MELTING AT A TEMPERATURE IN THE RANGE OF 200-201* C. WITH DECOMPOSITION; HAVING A DEGREE OF OPTICAL ROTATION (A)D25 OF -85.99* (1% IN CHLOROFORM); HAVING ANALYTICAL DATA: C, 65.66; H, 5.91; 0, 28, 43; HAVING VISIBLE AND ULTRA-VIOLET ABSORPTION SPECTRA IN ETHANOL SOLUTION EXHIBITING TWO MAXIMA AT 415 AND 286 MU; AND EXHIBITING CHARACTERISTIC ABSORPTIONS IN THE INFRARED REGION ON THE SPECTRUM IN POTASSIUM BROMIDE PELLET, AT THE FOLLOWING FREQUENCIES EXPRESSED IN RECIPROCAL CENTIMETERS: 3100, 2950, 1640, 1590, 1530, 1460, 1410, 1340, 1250, 1200, 1160, 1120, 1070, 1050, 1005, 960, 935, AND
 835. 